Raman detection of localized transferrin-coated gold nanoparticles inside a single cell

Abstract

We investigated the cellular uptake behavior of non-fluorescent metal nanoparticles (NPs) by use of surface-enhanced Raman scattering (SERS) combined with dark-field microscopy (DFM). The uptake of Au NPs inside a single cell could also be identified by DFM first and then confirmed by z-depth-dependent SERS at micrometer resolution. Guided by DFM for the location of Au NPs, an intracellular distribution assay was possible using Raman dyes with unique vibrational marker bands in order to identify the three-dimensional location inside the single cell by obtaining specific spectral features. Au NPs modified by 4-mercaptobenzoic acid (MBA) bearing its -COOH surface functional group were used to conjugate transferrin (Tf) protein using the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) reaction. The protein conjugation reaction on Au surfaces was examined by means of color change, absorption spectroscopy, and SERS. Our results demonstrate that DFM techniques combined with SERS may have great potential for monitoring biological processes with protein conjugation at the single-cell level.

Bioanalytical strategies for in-vitro and in-vivo evaluation of the toxicity induced by metallic nanoparticles

“…A mixture of glutaraldehyde and formaldehyde is also very commonly used as fixing agent. A fixing solution containing 3% paraformaldehyde and 0.5% glutaraldehyde was employed to visualize both the localization of SiO 2 NPs and Ag doped SiO 2 NPs in fibroblast-like fathead minnow cells [17], and Au NPs in human lung carcinoma cells A549 [37]. In some cases, resulting blocks embedded in resin are incubated in uranyl-acetate (1%-6%) to obtain a higher electron density and image contrast, which is especially useful for smaller particulate specimens.…”

“…If NPs are found in cells, their localization in different compartments such as endosomes, lysosomes, mitochondria, the nucleus or the citosol, may also provide some answers regarding their potential toxicity [16]. It is also of great importance the understanding of the complex processes that govern cellular uptake and intracellular fate of MNPs [17]. Finally, in vivo studies are also needed to design specific and safe clinical trials to develop new strategies for the applied use of MNPs and also to evaluate the risk to the environment [18].…”

Scanning angle Raman spectroscopy: Investigation of Raman scatter enhancement techniques for chemical analysis

“…Others have also combined SERS with dark field microscopy to provide information on the intracellular uptake mechanism and location in a single mammalian cell. 571 Itoh and co-workers used a combination of plasmon resonance Rayleigh scattering (PRRS) and SERS with Ag nanoparticles to measure mannoproteins on yeast cell walls.…”

Advanced Optical Imaging of Endocytosis

“…Toward similar goals of characterizing the endocytotic uptake and trafficking of gold nanoparticles for applications in biomedical diagnostics and targeted gene/drug delivery, Park et al used surface-enhanced Raman scattering (SERS) and dark field microscopy to visualize gold nanoparticles conjugated to transferrin protein (Park, et al, 2011). This work demonstrates the additional sensitivity offered by SERS over traditional Raman spectroscopy, and the potential of this method to not only follow nanoparticle distribution in three dimensions in a single living cell, but also make use of SERS spectral changes to indicate alterations in protein conjugation due to biochemical reactions.…”